Sign up to receive free email alerts when patent applications with chosen keywords are publishedSIGN UP

Abstract:

The invention relates to improvements in the field of drug delivery. More
particularly, the invention relates to polypeptides derived from
aprotinin and from aprotinin analogs as well as conjugates and
pharmaceutical compositions comprising these polypeptides or conjugates.
The present invention also relates to the use of these polypeptides for
transporting a compound or drug across the blood-brain barrier of a
mammal and in the treatment and diagnosis of neurological diseases.

Claims:

1. A method for treating a mammal having a neurological disease, said
method comprising administering to said mammal a conjugate comprising:
(a) a polypeptide comprising an amino acid sequence having at least 80%
identity to the sequence of Angiopep-1 (SEQ ID NO:67), wherein said
polypeptide has a substitution at the position corresponding to the
cysteine at position 7 of the Angiopep-1 sequence; and (b) an agent for
treating said neurological disease, wherein said agent is conjugated to
said polypeptide, wherein said conjugate is capable of crossing the
blood-brain barrier or an in vitro model thereof.

2. The method of claim 1, wherein said neurological disease is selected
from the group consisting of a brain cancer, a brain metastasis,
schizophrenia, epilepsy, Alzheimer's disease, Parkinson's disease,
Huntington's disease, stroke, and obesity.

3. The method of claim 2, wherein said brain cancer is a glioma.

4. The method of claim 3, wherein said glioma is a glioblastoma.

5. The method of claim 1, wherein said percent identity is at least 85%.

6. The method of claim 5, wherein said percent identity is at least 90%.

7. The method of claim 1, wherein said substitution is a serine
substitution.

9. The method of claim 7, wherein said polypeptide consists of the amino
acid sequence of Angiopep-2 (SEQ ID NO:97).

10. The method of claim 1, wherein said agent is an anticancer drug.

11. The method of claim 10, wherein said anticancer drug is selected from
the group consisting of paclitaxel, vinblastine, vincristine, etoposide,
doxorubicin, cyclophosphamide, taxotere, melphalan, and chlorambucil, or
a combination thereof.

12. The method of claim 11, wherein said anticancer drug is paclitaxel.

13. The method of claim 11, wherein said anticancer drug is etoposide.

14. The method of claim 11, wherein said anticancer drug is doxorubicin.

15. The method of claim 1, wherein said agent is a small molecule drug,
an antibiotic, a cellular toxin, an anti-angiogenic compound, or a
polypeptide.

16. The method of claim 15, wherein said agent is a chemotherapeutic
agent.

19. The method of claim 1, wherein said administration is intra-arterial,
intra-nasal, intra-peritoneal, intravenous, intramuscular, subcutaneous,
transdermal, or per os.

20. A method for transporting an agent across the blood-brain barrier of
a mammal, said method comprising administering to said mammal a conjugate
comprising: (a) a polypeptide comprising an amino acid sequence having at
least 80% identity to the sequence of Angiopep-1 (SEQ ID NO:67), wherein
said polypeptide has a substitution at the position corresponding to the
cysteine at position 7 of the Angiopep-1 sequence; and (b) said agent,
wherein said agent is conjugated to said polypeptide, wherein said
conjugate is capable of crossing the blood-brain barrier or an in vitro
model thereof.

21. The method of claim 20, wherein said percent identity is at least
85%.

22. The method of claim 21, wherein said percent identity is at least
90%.

23. The method of claim 20, wherein said substitution is a serine
substitution.

25. The method of claim 24, wherein said polypeptide consists of the
amino acid sequence of Angiopep-2 (SEQ ID NO:97).

26. The method of claim 20, wherein said agent is an anticancer drug.

27. The method of claim 26, wherein said anticancer drug is selected from
the group consisting of paclitaxel, vinblastine, vincristine, etoposide,
doxorubicin, cyclophosphamide, taxotere, melphalan, and chlorambucil, or
a combination thereof.

28. The method of claim 27, wherein said anticancer drug is paclitaxel.

29. The method of claim 27, wherein said anticancer drug is etoposide.

30. The method of claim 27, wherein said anticancer drug is doxorubicin.

31. The method of claim 20, wherein said agent is a small molecule drug,
an antibiotic, a cellular toxin, an anti-angiogenic compound, a
polypeptide, or a detectable label.

32. The method of claim 31, wherein said agent is a chemotherapeutic
agent.

36. The method of claim 35, wherein said detectable label is selected
from the group consisting of a radioimaging agent, an isotope, a
fluorescent label, a nuclear magnetic resonance label, a luminescent
label, a chromophore, a chemiluminescent label, or an enzymatic label.

37. The method of claim 36, wherein said detectable label is selected
from the group consisting of a green fluoresence protein, a histag,
β-galactosidase, indium-11, technitium-99, or iodine-131,
peroxidase, and phosphatase.

38. The method of claim 20, wherein said administration is
intra-arterial, intra-nasal, intra-peritoneal, intravenous,
intramuscular, subcutaneous, transdermal, or per os.

39. A method of treating brain cancer, said method comprising
administering to said patient a conjugate comprising: (a) a polypeptide
comprising Angiopep-2 (SEQ ID NO:97); and (b) an anticancer cancer agent,
wherein said agent is conjugated to said Angiopep-2.

40. The method of claim 39, wherein said anticancer agent is paclitaxel.

41. The method of claim 40, wherein said conjugate comprises: (a) a
polypeptide consisting of Angiopep-2; and (b) three paclitaxel molecules,
wherein said paclitaxel molecules are each conjugated to said Angiopep-2.

42. The method of claim 41, wherein each of said paclitaxel molecules is
conjugated through a linker that joins the hydroxyl on the 2' carbon of
paclitaxel to an amine group on said Angiopep-2, said linker having the
following structure: ##STR00001##

43. The method of claim 42, wherein said administration is intravenous.

44. The method of claim 39, wherein said anticancer agent is etoposide.

45. The method of claim 39, wherein said anticancer agent is doxorubicin.

46. The method of claim 1, wherein the agent is an antibody or a
biologically active fragment thereof.

47. The method of claim 46, wherein the fragment is an Fv fragment, an
F(ab)2 fragment, an F(ab)2' fragment, or an Fab fragment.

48. The method of claim 20, wherein the agent is an antibody or a
biologically active fragment thereof.

49. The method of claim 48, wherein the fragment is an Fv fragment, an
F(ab)2 fragment, an F(ab)2' fragment, or an Fab fragment.

Description:

FIELD OF THE INVENTION

[0001] The present invention relates to improvements in the field of drug
delivery. More particularly, the invention relates to polypeptides,
conjugates and pharmaceutical compositions comprising the polypeptides or
conjugates of the present invention. The present invention also relates
to the use of these polypeptides and conjugates for transporting a
compound or drug across the blood-brain barrier of a mammal and in the
treatment and diagnosis of neurological diseases.

BACKGROUND OF THE INVENTION

[0002] In the development of a new therapy for brain pathologies, the
blood-brain barrier (BBB) is considered as a major obstacle for the
potential use of drugs for treating disorders of the central nervous
system (CNS). The global market for CNS drugs was $33 billion in 1998,
which was roughly half that of global market for cardiovascular drugs,
even though in the United States, nearly twice as many people suffer from
CNS disorders as from cardiovascular diseases. The reason for this
lopsidedness is that more than 98% of all potential CNS drugs do not
cross the blood-brain barrier. In addition, more than 99% of worldwide
CNS drug development is devoted solely to CNS drug discovery, and less
than 1% is directed to CNS drug delivery. This ratio could explain why no
efficient treatment is currently available for the major neurological
diseases such as brain tumors, Alzheimer's and stroke.

[0003] The brain is shielded against potentially toxic substances by the
presence of two barrier systems: the blood-brain barrier (BBB) and the
blood-cerebrospinal fluid barrier (BCSFB). The BBB is considered to be
the major route for the uptake of serum ligands since its surface area is
approximately 5000-fold greater than that of BCSFB. The brain
endothelium, which constitutes the BBB, represents the major obstacle for
the use of potential drugs against many disorders of the CNS.

[0004] As a general rule, only lipophilic molecules smaller than about 500
Daltons may pass across the BBB, i.e., from blood to brain. However, the
size of many drugs that show promising results in animal studies for
treating CNS disorders is considerably bigger. Thus, peptide and protein
therapeutics are generally excluded from transport from blood to brain,
owing to the negligible permeability of the brain capillary endothelial
wall to these drugs. Brain capillary endothelial cells (BCECs) are
closely sealed by tight junctions, possess few fenestrae and few
endocytic vesicles as compared to capillaries of other organs. BCECs are
surrounded by extracellular matrix, astrocytes, pericytes and microglial
cells. The close association of endothelial cells with the astrocyte foot
processes and the basement membrane of capillaries are important for the
development and maintenance of the BBB properties that permit tight
control of blood-brain exchange.

[0005] International publication WO2004/060403 discloses an invention made
by the inventors relating to molecules for transporting a drug across the
blood brain barrier. Otherwise, to date, there is no efficient drug
delivery approach available for the brain. Methods under investigation
for peptide and protein drug delivery to the brain may be divided in
three principal strategies. Firstly, invasive procedures include the
direct intraventricular administration of drugs by means of surgery, and
the temporary disruption of the BBB via intracarotid infusion of
hyperosmolar solutions. Secondly, the pharmacologically-based strategy
consists in facilitating the passage through the BBB by increasing the
lipid solubility of peptides or proteins. Thirdly, physiologic-based
strategies exploit the various carrier mechanisms at the BBB, which have
been characterized in the recent years. In this approach, drugs are
attached to a protein vector that performs like receptors-targeted
delivery vehicle on the BBB. This approach is highly specific and
presents high efficacy with an extreme flexibility for clinical
indications with unlimited targets. The latter approach has been, and is
still, investigated by the inventors, who came up with the molecules
described in the afore-mentioned publication and those of the present
invention.

[0006] U.S. Pat. No. 5,807,980 describes Bovine Pancreatic Trypsin
Inhibitor (aprotinin)-derived inhibitors as well as a method for their
preparation and therapeutic use. These peptides are used for the
treatment of a condition characterized by an abnormal appearance or
amount of tissue factor and/or factor VIIIa such as abnormal thrombosis.

[0009] It would be highly desirable to be provided with improved molecules
that can act as carriers or vectors for transporting a compound or drug
across the BBB of an individual.

SUMMARY OF THE INVENTION

[0010] One aim of the present invention is to provide an improvement in
the field of drug delivery.

[0011] Another aim of the present invention is to provide a non-invasive
and flexible method and carrier for transporting a compound or drug
across the blood-brain barrier of an individual.

[0012] The present application discloses new molecules which may be able,
for example, of transporting desirable compounds across the blood brain
barrier.

[0013] In a first aspect the present invention provides a biologically
active polypeptide which may be able to cross (i.e., crossing) a cell
layer mimicking (which mimics) a mammalian blood brain barrier in an in
vitro assay, the polypeptide may be selected, for example, from the group
of [0014] aprotinin (SEQ ID NO.:98), [0015] an aprotinin analogue
[0016] an aprotinin fragment which may comprise (or may consist
essentially of) the amino acid sequence defined in SEQ ID NO.:1, [0017] a
biologically active analogue of SEQ ID NO.:1, [0018] a biologically
active fragment of SEQ ID NO.:1, and; [0019] a biologically active
fragment of a SEQ ID NO.:1 analogue.

[0020] In a second aspect the present invention provides, a biologically
active polypeptide which may be able to cross (i.e., crossing) a cell
layer mimicking (which mimics) a mammalian blood brain barrier in an in
vitro assay, the polypeptide may be selected, for example, from the group
of; [0021] an aprotinin fragment which may comprise the amino acid
sequence defined in SEQ ID NO.:1, [0022] a biologically active analogue
of SEQ ID NO.:1, [0023] a biologically active fragment of SEQ ID NO.:1
and; [0024] a biologically active fragment of a SEQ ID NO.:1 analogue.

[0025] In accordance with the present invention the aprotinin fragment may
consist of the sequence defined in SEQ ID NO.:1. Further in accordance
with the present invention, the aprotinin fragment may comprise SEQ ID
NO.1 and may have a length of from about 19 amino acids to about 54 amino
acids, e.g., from 10 to 50 amino acids in length, from 10 to 30 amino
acids in length etc.

[0026] In accordance with the present invention, the biologically active
analogue of SEQ ID NO.:1, may have a length of from about 19 amino acids
to about 54 amino acids (e.g., including for example 21 to 23, 25 to 34,
36 to 50 and 52 to 54), or of from about 19 amino acids to about 50 amino
acids, or from about 19 amino acids to about 34 amino acids (e.g., 19,
20, 21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34), of from about 19
amino acids to about 23 amino acids or of about 19, 20, 21, 22, 23, 24,
35, 51, amino acids.

[0027] A biologically active fragment of a polypeptide (e.g., of 19 amino
acids) described herein may include for example a polypeptide of from
about 7, 8, 9 or 10 to 18 amino acids. Therefore, in accordance with the
present invention, a biologically active fragment of SEQ ID NO.:1 or of a
SEQ ID NO.:1 analogue may have a length of from about 7 to about 18 amino
acids or from about 10 about 18.

[0028] U.S. Pat. No. 5,807,980 describes a polypeptide which is identified
herein as SEQ ID NO.:102.

[0029] U.S. Pat. No. 5,780,265 describes a polypeptide which is identified
herein as SEQ ID NO.:103.

[0030] The aprotinin amino acid sequence (SEQ ID NO.:98), the Angiopep-1
amino acid sequence (SEQ ID NO.:67), as well as some sequences of
biologically active analogs may be found for example in international
application no. PCT/CA2004/000011 published on Jul. 22, 2004 in under
international publication no. WO2004/060403. Additionally, international
publication No. WO04/060403 describes a polypeptide which is identified
herein as SEQ ID NO.: 104.

[0032] Examples of aprotinin analogs may be found by performing a protein
blast (Genebank: www.ncbi.nlm.nih.gov/BLAST/) of the synthetic aprotinin
sequence (or portion thereof) disclosed in international application no.
PCT/CA2004/000011. Exemplary aprotinin analogs may be found, for example
under accession nos. CAA37967 (GI:58005), 1405218C (GI:3604747) etc.

[0033] In a further aspect the present invention provides a biologically
active polypeptide which may be able to cross (i.e., crossing) a cell
layer mimicking (which mimics) a mammalian blood brain barrier in an in
vitro assay, the polypeptide may be selected, for example, from the group
of; [0034] an aprotinin fragment of from 19 to 54 (e.g., 19-50) amino
acid long, which may comprise SEQ ID NO.:1, [0035] an aprotinin fragment
consisting of SEQ ID NO.:1, [0036] a biologically active analogue of SEQ
ID NO.:1 of from about 19 to 50 amino acids long, and; [0037] a
biologically active fragment of SEQ ID NO.:1 (of from 10 to 18 amino
acids) or biologically active fragment of a SEQ ID NO.:1 analogue (of
from about 10 to 18 amino acids).

[0038] In accordance with the present invention there is provided a
biologically active analogue of SEQ ID NO.:1 which may be selected, for
example, from the group consisting of [0039] a SEQ ID NO.:1 analogue
which may comprise at least 35% identity with the amino acid sequence of
SEQ ID NO.:1, [0040] a SEQ ID NO.:1 analogue which may comprise at least
40% identity with the amino acid sequence of SEQ ID NO.:1, [0041] a SEQ
ID NO.:1 analogue which may comprise at least 50° A) identity with
the amino acid sequence of SEQ ID NO.:1, [0042] a SEQ ID NO.:1 analogue
which may comprise at least 60% identity with the amino acid sequence of
SEQ ID NO.:1, [0043] a SEQ ID NO.:1 analogue which may comprise at least
70% identity with the amino acid sequence of SEQ ID NO.:1, [0044] a SEQ
ID NO.:1 analogue which may comprise at least 80% identity with the amino
acid sequence of SEQ ID NO.:1, [0045] a SEQ ID NO.:1 analogue which may
comprise at least 90% identity with the amino acid sequence of SEQ ID
NO.:1 and; [0046] a SEQ ID NO.:1 analogue which may comprise at least 95%
(i.e., 96%, 97%, 98%, 99% and 100%) identity with the amino acid sequence
of SEQ ID NO.:1.

[0047] For example, the biologically active analogue of SEQ ID NO.:1 may
comprise an amino acid sequence selected from the group consisting of an
amino acid sequence defined in any one of SEQ ID NO.:2 to SEQ ID NO.: 62,
SEQ ID NO.: 68 to SEQ ID NO.: 93, and SEQ ID NO.:97 as well as 99, 100
and 101. When the polypeptide of the present invention comprises, for
example, SEQ ID NO.:99, 100 or 101, the polypeptide may have an amino
acid sequence of from about 10 to 50 amino acids, e.g., from 10 to 30
amino acids in length.

[0048] Further in accordance with the present invention, the biologically
active analogue of SEQ ID NO.:1 may comprise the amino acid sequence
defined in SEQ ID NO.:67 (i.e., polypeptide no. 67 which is an amidated
version of SEQ ID NO.:67 (Angiopep-1)).

[0049] The polypeptides of the present invention may be amidated, i.e.,
may have an amidated amino acid sequence. For example, the polypeptide of
SEQ ID NO.:67 may be amidated (polypeptide no. 67).

[0050] Portion of the present invention may relate to the polypeptides
defined herein with the exception of polypeptides defined in SEQ ID NO.:
102, 103, 104 and 105, while other portion of the invention may include
these peptides. For example and without limitation, conjugates comprising
these peptides as well as their use for treating a neurological disease
(e.g., brain tumor), method of treatment of a neurological disease (e.g.,
brain tumor), pharmaceutical composition for treating a neurological
disease, etc. are encompassed by the present invention.

[0051] In yet a further aspect the present invention provides a
biologically active polypeptide which may be able to cross (i.e.,
crossing) a cell layer mimicking (which mimics) a mammalian blood brain
barrier in an in vitro assay, the polypeptide may be selected, for
example, from the group of; [0052] an aprotinin fragment of from 19 to
54 (e.g., 19-50) amino acid long, which may comprise SEQ ID NO.:1, [0053]
an aprotinin fragment consisting of SEQ ID NO.:1, [0054] a biologically
active analogue of SEQ ID NO.:1 of from about 19 to 50 amino acids long,
provided that said analogue does not comprise SEQ ID NO.: 102, 103, 104
or 105 and provided that when said analogue consists of SEQ ID NO.:67
said analogue is amidated, [0055] a biologically active fragment of SEQ
ID NO.:1 of from 10 to 18 amino acids, and; [0056] a biologically active
fragment of a SEQ ID NO.:1 analogue of from about 10 to 18 amino acids.

[0057] Further in accordance with the present invention, the biologically
active fragment of SEQ ID NO.:1 or the biologically active fragment of a
SEQ ID NO.:1 analogue may comprise at least 9 or at least 10 (consecutive
or contiguous) amino acids of SEQ ID NO.1 or of the SEQ ID NO.:1
analogue.

[0058] The polypeptides of the present invention may have an amino acid
sequence which may comprise of from between 1 to 12 amino acid
substitutions (i.e., SEQ ID NO.:91). For example, the amino acid
substitution may be from between 1 to 10 amino acid substitutions, or
from 1 to 5 amino acid substitutions. In accordance with the present
invention, the amino acid substitution may be a non-conservative amino
acid substitution or a conservative amino acid substitution.

[0059] For example, when a polypeptide of the present invention comprises
amino acids which are identical to those of SEQ ID NO.:1 and other amino
acids which are not identical (non-identical), those which are
non-identical may be a conservative amino acid substitution. The
comparison of identical and non-identical amino acids may be performed by
looking at a corresponding location.

[0063] In accordance, with the present invention, the carrier may more
particularly be selected from the group consisting of peptide Nos. 5, 67,
76, 91 and peptide 97 (i.e., SEQ ID NO.:5, 67, 76, 91 and 97
(Angiopep-2)). The carrier may be used, for example, for transporting an
agent attached thereto across a blood-brain barrier. In accordance with
the present invention, the carrier may be able to cross the blood-brain
barrier after attachment to the agent and may therefore be able to
transport the agent across the blood-brain barrier.

[0064] In accordance with the present invention, the polypeptides may be
in an isolated form or in a substantially purified form.

[0065] More particularly, the present invention provides a carrier for
transporting an agent attached thereto across a blood-brain barrier,
wherein the carrier may be able to cross the blood-brain barrier after
attachment to the agent and thereby transport the agent across the
blood-brain barrier. The carrier may comprise at least one polypeptide of
the present invention (provided that when said polypeptide or carrier
consist of SEQ ID NO.:67, said polypeptide is modified by a group e.g.,
amidated). For example, the carrier may be selected from a class of
molecules related to aprotinin.

[0066] The transporting activity which is effected by the carrier does not
affect blood-brain barrier integrity. The transporting of an agent may
result, for example, in the delivery of the agent to the central nervous
system (CNS) of an individual.

[0067] It is to be understood herein that the polypeptides of the present
invention may be synthesized chemically (e.g., solid phase synthesis) or
may be produced by recombinant DNA technology. Codons which encode
specific amino acids are well known in the art and is discuss, for
example, in Biochemistry (third edition; 1988, Lubert Stryer, Stanford
University, W.H. Freeman and Company, New-York). A nucleotide sequence
encoding a carrier of the present invention is therefore encompassed
herein. More particularly, nucleotide sequences (deoxyribonucleotides or
ribonucleotides or derivatives thereof) encoding a polypeptide selected
from the group consisting of any one of SEQ ID NO.:1 to 97, are
encompassed by the present invention. An exemplary nucleotide sequence
encoding an aprotinin analogue is illustrated in SEQ ID NO.:106 and may
be found in Gene Bank under accession no. X04666. This sequence encodes
an aprotinin analogue having a lysine at position 16 (with reference to
the amino acid sequence encoded by SEQ ID NO.:106) instead of a valine as
found in SEQ ID NO.:98. A mutation in the nucleotide sequence of SEQ ID
NO.:106 may be introduced by methods known in the art to change the
produce the peptide of SEQ ID NO.:98 having a valine in position 16.
Techniques known in the art may be used to introduce further mutations in
the nucleotide sequence to encode analogues of the present invention.
Fragments may be obtained from this nucleotide sequence by enzymatic
digestion or polymerase chain reaction, etc. Alternatively, a desired
nucleotide sequence may be synthesized chemically by methods known in the
art.

[0068] In a further aspect, the present invention relates to a conjugate
which may comprise a carrier selected from the group consisting of any
one of the polypeptide of the present invention, and an agent selected
from the group consisting, for example, of a drug (e.g., a small molecule
drug, e.g., an antibiotic), a medicine, a detectable label, a protein
(e.g., an enzyme), protein-based compound (e.g., a protein complex
comprising one or polypeptide chain) and a polypeptide (peptide). The
agent may be more particularly, a molecule which is active at the level
of the central nervous system. The agent may be any agent for treating or
detecting a neurological disease.

[0069] In accordance with the present invention the carrier which is part
of conjugate may be selected, for example, from the group of; [0070] an
aprotinin fragment of from 10 to 54 (e.g., 19-50) amino acid long, which
may comprise SEQ ID NO.:1, [0071] an aprotinin fragment consisting of SEQ
ID NO.:1, [0072] a biologically active analogue of SEQ ID NO.:1 (e.g., of
from about 19 to 50 amino acids long), provided that when said analogue
consists of SEQ ID NO.:67 said analogue is amidated, [0073] a
biologically active fragment of SEQ ID NO.:1 of from 10 to 18 amino
acids, and; [0074] biologically active fragment of a SEQ ID NO.:1
analogue of from about 10 to 18 amino acids.

[0075] In accordance with the present invention, the agent may have a
maximum molecular weight of about 160,000 Daltons.

[0076] Further in accordance with the present invention, the transporting
activity may be effected by receptor-mediated transcytosis or
adsorptive-mediated transcytosis. The agent may be one able to be
transported by such mechanism.

[0077] Further in accordance with the present invention, the conjugate may
be in the form of a fusion protein which may have a first moiety
consisting essentially of the carrier of the present invention and a
second moiety consisting essentially of a protein or protein-based agent.

[0078] Exemplary neurological diseases which may be treated or detected by
the carrier and/or conjugate is a disease selected, for example, from the
group consisting of a brain tumor, a brain metastasis, schizophrenia,
epilepsy, Alzheimer's disease, Parkinson's disease, Huntington's disease,
stroke and blood-brain barrier related malfunctions (e.g., obesity).

[0079] In accordance with the present invention, the blood-brain barrier
related malfunction is obesity. Also in accordance with the present
invention, the agent which may be conjugated with the carrier of the
present invention may be a leptin. A conjugate comprising a leptin and a
carrier may be used, for example, in the treatment of obesity.

[0080] In accordance with the present invention, the detectable label may
be a radioimaging agent. Example of a label which may be conjugated with
the carrier of the present invention and which is encompassed herein
includes, for example and without limitation, an isotope, a fluorescent
label (e.g., rhodamine), a reporter molecule (e.g., biotin), etc. Other
examples of detectable labels include, for example, a green fluorescent
protein, biotin, a histag protein and β-galactosidase.

[0082] Also in accordance with the present invention, the agent may be a
small molecule drug such as an anticancer drug (e.g., for treating a
brain tumor). An anticancer drug encompassed by the present invention may
include, for example, a drug having a group allowing it's conjugation to
the carrier of the present invention. Examples of anticancer drug
includes, for example, without limitation, a drug which may be selected
from the group consisting of paclitaxel (Taxol), vinblastine,
vincristine, etoposide, doxorubicin, cyclophosphamide, taxotere,
melphalan, chlorambucil, and any combination.

[0083] More particularly, the conjugate of the present invention may
comprise the formula R-L-M or pharmaceutically acceptable salts thereof,
wherein R is a class of molecules related to aprotinin (e.g., aprotinin,
aprotinin fragment, Angiopep-1, Angiopep-2, analogs, derivatives or
fragments). For example, R may be a carrier selected from a class of
molecules related to aprotinin able to cross the blood-brain barrier
after attachment to L-M and thereby transport M across the blood-brain
barrier. L may be a linker or a bond (chemical bond). M may be an agent
selected from the group consisting of a drug (e.g., a small molecule
drug), a medicine, a (detectable) label, a protein or protein-based
compound (e.g., antibody, an antibody fragment), an antibiotic, an
anti-cancer agent, an anti-angiogenic compound and a polypeptide or any
molecule active at the level of the central nervous system. It is to be
understood herein that the formula R-L-M is not intended to be restricted
to a specific order or specific ratio. As being exemplified herein, M may
be found in several ratios over R.

[0084] For example, conjugates of formula R-L-M or a pharmaceutically
acceptable salt thereof, may be used for transporting M across a
blood-brain barrier, where R may be for example, a carrier selected from
the group consisting of peptide Nos: 5, 67, 76, 91 and 97 as described in
herein. The carrier may be able to cross the blood-brain barrier after
attachment to L-M and may therefore transport M across the blood-brain
barrier.

[0085] In accordance with the present invention, M may be an agent useful
for treating or diagnosing a neurological disease.

[0086] It is to be understood herein that when more than one carrier
conjugation site are available or present, more than one drug or drug
molecule may be conjugated to the carrier of the present invention.
Therefore, the conjugate may comprise one or more drug molecules. The
conjugate may be active by itself, i.e., the drug may be active even when
associated with the carrier. Also in accordance with the present
invention, the compound may or may not be released from the carrier i.e.,
generally after transport across the blood-brain barrier. The compound
may therefore be releasable from the conjugate (or from the carrier) and
may become active thereafter. More particularly, the agent may be
releasable from the carrier after transport across the blood-brain
barrier.

[0087] In accordance with another embodiment of the present invention,
there is provided a conjugate for transporting an agent across a
blood-brain barrier, the conjugate may comprise: (a) a carrier; and (b)
an agent attached to the carrier, wherein the conjugate is able to cross
the blood-brain barrier and thereby transport the agent across the
blood-brain barrier.

[0088] In a further aspect, the present invention relates to the use of a
carrier or a conjugate of the present invention for transporting an agent
across a blood brain barrier of a mammal in need thereof.

[0089] In yet a further aspect, the present invention relates to the use
of a class of molecules related to aprotinin for transporting a compound
attached thereto across the blood-brain barrier of a patient.

[0090] In an additional aspect, the present invention relates to the use
of a carrier or a conjugate as described herein for the diagnosis of a
neurological disease or a central nervous system disease. For example,
the carrier or conjugate may be used for the in vivo detection of a
neurological disease.

[0091] The carrier may be selected, for example, from the group of
(biologically active); [0092] aprotinin (SEQ ID NO.:98), [0093] an
aprotinin fragment which may comprise the amino acid sequence defined in
SEQ ID NO.:1, [0094] an aprotinin fragment consisting of SEQ ID NO.:1,
[0095] a biologically active analogue of SEQ ID NO.:1, and; [0096] a
biologically active fragment of SEQ ID NO.:1 or biologically active
fragment of a SEQ ID NO.:1 analogue.

[0097] More particularly, the carrier may be selected, for example, from
the group of (biologically active); [0098] an aprotinin fragment which
may comprise the amino acid sequence defined in SEQ ID NO.:1, [0099] an
aprotinin fragment consisting of SEQ ID NO.:1, [0100] a biologically
active analogue of SEQ ID NO.:1, and; [0101] a biologically active
fragment of SEQ ID NO.:1 or biologically active fragment of a SEQ ID
NO.:1 analogue.

[0102] In accordance with the present invention, when that analogue
consists of SEQ ID NO.:67, said analogue is amidated.

[0103] Even more particularly, the carrier may be selected, for example,
from the group of; [0104] an aprotinin fragment of from 10 to 54 (e.g.,
19-50) amino acid long, which may comprise SEQ ID NO.:1, [0105] an
aprotinin fragment consisting of SEQ ID NO.:1, [0106] a biologically
active analogue of SEQ ID NO.:1 (e.g., of from about 19 to 50 amino acids
long), provided that when said analogue consists of SEQ ID NO.:67, said
analogue is amidated, [0107] a biologically active fragment of SEQ ID
NO.:1 of from 10 to 18 amino acids, and; [0108] a biologically active
fragment of a SEQ ID NO.:1 analogue of from about 10 to 18 amino acids.

[0109] In another aspect, the present invention relates to the use of a
class of molecules related to aprotinin in the manufacture of a
medicament.

[0110] According to the present invention, there is provided the use of a
class of molecules related to aprotinin in the manufacture of a
medicament for treating a neurological disease, or for treating a central
nervous system disorder.

[0111] In yet another aspect, the present invention relate to the use of a
carrier or conjugate described herein, in the manufacture of a medicament
for treating a brain disease (a brain-associated disease) or neurological
disease, for the diagnosis of a brain disease or neurological disease or
for transporting an agent across the blood-brain barrier

[0112] In an additional aspect, the present invention relates to the use
of a carrier or conjugate of the present invention for treating a mammal
having, for example, a neurological disease or for the diagnosis of a
neurological disease in a mammal in need thereof.

[0114] In a further aspect, the present invention relates to a method for
transporting an agent across the blood-brain barrier of a mammal (human,
animal), which may comprise the step of administering to the mammal a
compound comprising the agent attached to a class of molecules related to
aprotinin.

[0115] In yet a further aspect, the present invention provides a method
for treating a neurological disease of a patient comprising administering
to the patient a medicament comprising a class of molecules related to
aprotinin, and a compound adapted to treat the disease, the compound
being attached to the class of molecules related to aprotinin.

[0116] In an additional aspect, there is provided a method for treating a
central nervous system disorder of a patient comprising administering to
the patient a medicament comprising a class of molecules related to
aprotinin, and a compound adapted to treat the disease, the compound
being attached to the aprotinin.

[0117] In yet an additional aspect there is provided a method for
transporting an agent across a blood-brain barrier, which comprises the
step of administering to an individual a pharmaceutical composition of
the present Invention.

[0118] The present invention also relates, in a further aspect to a method
for treating a mammal (e.g., a patient) in need thereof (e.g., a patient
having a neurological disease). The method may comprise administering a
carrier, a conjugate and/or a pharmaceutical composition of the present
invention to the mammal.

[0119] The present invention additionally relates to a method for (of)
diagnosing (i.e., a diagnostic method) a neurological disease in a mammal
(e.g., a patient) in need thereof. The method may comprise administering
a carrier, a conjugate and/or a pharmaceutical composition of the present
invention to the mammal (human individual, patient, animal).

[0120] In accordance with the present invention, the administration may be
performed intra-arterially, intra-nasally, intra-peritoneally,
intravenously, intramuscularly, subcutaneously, transdermally or per os.

[0121] In accordance with the present invention, the pharmaceutical
composition may be administered to the mammal in a therapeutically
effective amount.

[0122] A mammal in need (individual in need) may be, for example, a mammal
I which has or is at risk of having a neurological disease, a central
nervous system disease, brain cancer, a brain metastasis, etc.

[0123] In an additional aspect, the present invention relates to a
pharmaceutical composition which may comprise, for example; [0124] a
carrier (which may be selected from the group consisting of any of the
polypeptide described herein) or conjugate of the present invention; and
[0125] a pharmaceutically acceptable carrier, e.g., a pharmaceutically
acceptable excipient.

[0126] In accordance with the present invention, the pharmaceutical
composition may be used, for example, for the treatment of a neurological
disease.

[0127] Further in accordance with the present invention, the
pharmaceutical composition may be used, for example, for the diagnosis of
a neurological disease.

[0128] Also in accordance with the present invention, the pharmaceutical
composition may be used for example, for transporting an agent across a
blood-brain barrier.

[0129] Also in accordance with the present invention, the pharmaceutical
composition may be used for example, for the delivery of an agent to the
CNS of an individual.

[0130] Further in accordance with the present invention, the
pharmaceutical composition may be used for example, for treating a
central nervous system disorder of a mammal in need thereof.

[0131] In accordance with the present invention, pharmaceutical
composition may be used for delivery of an agent to the CNS of an
individual

[0132] It is to be understood herein that a pharmaceutically acceptable
salts of a carrier (polypeptide) or of a conjugate is encompassed by the
present invention.

[0133] The composition (pharmaceutical composition) may thus comprise a
medicament manufactured as defined herein in association with a
pharmaceutically acceptable excipient.

[0134] For the purpose of the present invention the following terms are
defined below.

[0135] The term "carrier" or "vector" is intended to mean a compound or
molecule such as a polypeptide that is able to transport a compound. For
example, transport may occur across the blood-brain barrier. The carrier
may be attached to (covalently or not) or conjugated to another compound
or agent and thereby may be able to transport the other compound or agent
across the blood-brain barrier. For example, the carrier may bind to
receptors present on brain endothelial cells and thereby be transported
across the blood-brain barrier by transcytosis. The carrier may be a
molecule for which high levels of transendothelial transport may be
obtained, without affecting the blood-brain barrier integrity. The
carrier may be, but is not limited to, a protein, a peptide or a
peptidomimetic and may be naturally occurring or produced by chemical
synthesis or recombinant genetic technology (genetic engineering).

[0136] The term "conjugate" is intended to mean a combination of a carrier
and another compound or agent. The conjugation may be chemical in nature,
such as via a linker, or genetic in nature for example by recombinant
genetic technology, such as in a fusion protein with for example a
reporter molecule (e.g. green fluorescent protein, β-galactosidase,
Histag, etc.).

[0137] The expression "small molecule drug" is intended to mean a drug
having a molecular weight of 1000 g/mol or less.

[0138] The terms "treatment", "treating" and the like are intended to mean
obtaining a desired pharmacologic and/or physiologic effect, e.g.,
inhibition of cancer cell growth, death of a cancer cell or amelioration
of a neurological disease or condition. The effect may be prophylactic in
terms of completely or partially preventing a disease or symptom thereof
and/or may be therapeutic in terms of a partial or complete cure for a
disease and/or adverse effect attributable to the disease. "Treatment" as
used herein covers any treatment of a disease in a mammal, particularly a
human, and includes: (a) preventing a disease or condition (e.g.,
preventing cancer) from occurring in an individual who may be predisposed
to the disease but has not yet been diagnosed as having it; (b)
inhibiting a disease, (e.g., arresting its development); or (c) relieving
a disease (e.g., reducing symptoms associated with a disease).
"Treatment" as used herein covers any administration of a pharmaceutical
agent or compound to an individual to treat, cure, alleviate, improve,
diminish or inhibit a condition in the individual, including, without
limitation, administering a carrier-agent conjugate to an individual.

[0139] The term "cancer" is intended to mean any cellular malignancy whose
unique trait is the loss of normal controls which results in unregulated
growth, lack of differentiation and ability to invade local tissues and
metastasize. Cancer can develop in any tissue of any organ. More
specifically, cancer is intended to include, without limitation, cancer
of the brain.

[0140] The term "administering" and "administration" is intended to mean a
mode of delivery including, without limitation, intra-arterially,
intra-nasally, intra-peritoneally, intravenously, intramuscularly,
sub-cutaneously, transdermally or per os. A daily dosage can be divided
into one, two or more doses in a suitable form to be administered at one,
two or more times throughout a time period.

[0141] The term "therapeutically effective" or "effective amount" is
intended to mean an amount of a compound sufficient to substantially
improve some symptom associated with a disease or a medical condition.
For example, in the treatment of cancer or a mental condition or
neurological or CNS disease, an agent or compound which decreases,
prevents, delays, suppresses, or arrests any symptom of the disease or
condition would be therapeutically effective. A therapeutically effective
amount of an agent or compound is not required to cure a disease or
condition but will provide a treatment for a disease or condition such
that the onset of the disease or condition is delayed, hindered, or
prevented, or the disease or condition symptoms are ameliorated, or the
term of the disease or condition is changed or, for example, is less
severe or recovery is accelerated in an individual.

[0142] The carrier and conjugates of the present invention may be used in
combination with either conventional methods of treatment and/or therapy
or may be used separately from conventional methods of treatment and/or
therapy.

[0143] When the conjugates of this invention are administered in
combination therapies with other agents, they may be administered
sequentially or concurrently to an individual. Alternatively,
pharmaceutical compositions according to the present invention may be
comprised of a combination of a carrier-agent conjugate of the present
invention in association with a pharmaceutically acceptable excipient, as
described herein, and another therapeutic or prophylactic agent known in
the art.

[0144] Pharmaceutically acceptable acid addition salts may be prepared by
methods known and used in the art and are encompassed by the present
invention.

[0145] Biologically active polypeptides of the present invention encompass
functional derivatives. The term "functional derivative" is intended to
mean a "chemical derivative", "fragment", or "variant" biologically
active sequence or portion of a carrier or agent or conjugate and a salt
thereof of the present invention. A carrier functional derivative may be
able to be attached to or conjugated to another compound or agent and
cross the blood-brain barrier and thereby be able to transport the other
compound or agent across the blood-brain barrier.

[0146] The term "chemical derivative" is intended to mean a carrier, an
agent, or a conjugate of the present invention, which contains additional
chemical moieties not a part of the carrier, agent or carrier-agent
conjugate. Covalent modifications are included within the scope of this
invention. A chemical derivative may be conveniently prepared by direct
chemical synthesis, using methods well known in the art. Such
modifications may be, for example, introduced into a protein or peptide
carrier, agent or carrier-agent conjugate by reacting targeted amino acid
residues with an organic derivatizing agent that is capable of reacting
with selected side chains or terminal residues. A carrier chemical
derivative is able to cross the blood-brain barrier and be attached to or
conjugated to another compound or agent and thereby be able to transport
the other compound or agent across the blood-brain barrier. In a
preferred embodiment, very high levels of transendothelial transport
across the blood-brain barrier are obtained without any effects on the
blood-brain barrier integrity.

[0147] The term "agent" is intended to mean without distinction an
antibody, a drug (such as a medicinal drug) or a compound such as a
therapeutic agent or compound, a marker, a tracer or an imaging compound.

[0148] The term "therapeutic agent" or "agent" is intended to mean an
agent and/or medicine and/or drug used to treat the symptoms of a
disease, physical or mental condition, injury or infection and includes,
but is not limited to, antibiotics, anti-cancer agents, anti-angiogenic
agents and molecules active at the level of the central nervous system
Paclitaxel, for example, can be administered intravenously to treat brain
cancer.

[0149] The term "condition" is intended to mean any situation causing
pain, discomfort, sickness, disease or disability (mental or physical) to
or in an individual, including neurological disease, injury, infection,
or chronic or acute pain. Neurological diseases which can be treated with
the present invention include, but are not limited to, brain tumors,
brain metastases, schizophrenia, epilepsy, Alzheimer's disease,
Parkinson's disease, Huntington's disease and stroke.

[0151] Further, as used herein "pharmaceutically acceptable carrier" or
"pharmaceutical carrier" are known in the art and include, but are not
limited to, 0.01-0.1 M or 0.05 M phosphate buffer or 0.8% saline.
Additionally, such pharmaceutically acceptable carriers may be aqueous or
non-aqueous solutions, suspensions, and emulsions. Examples of
non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable
oils such as olive oil, and injectable organic esters such as ethyl
oleate. Aqueous carriers include water, alcoholic/aqueous solutions,
emulsions or suspensions, including saline and buffered media. Parenteral
vehicles include sodium chloride solution, Ringer's dextrose, dextrose
and sodium chloride, lactated Ringer's or fixed oils. Intravenous
vehicles include fluid and nutrient replenishers, electrolyte
replenishers such as those based on Ringer's dextrose, and the like.
Preservatives and other additives may also be present, such as, for
example, antimicrobials, antioxidants, collating agents, inert gases and
the like.

[0152] A "analogue" is to be understood herein as a polypeptide
originating from an original sequence or from a portion of an original
sequence and which may comprise one or more modification; for example,
one or more modification in the amino acid sequence (e.g., an amino acid
addition, deletion, insertion, substitution etc.), one or more
modification in the backbone or side-chain of one or more amino acid, or
an addition of a group or another molecule to one or more amino acids
(side-chains or backbone). An "analogue" is therefore understood herein
as a molecule having a biological activity and chemical structure (or a
portion of its structure) similar to that of a polypeptide described
herein. An analog comprises a polypeptide which may have, for example,
one or more amino acid insertion, either at one or both of the ends of
the polypeptide and/or inside the amino acid sequence of the polypeptide.

[0153] An "analogue" may have sequence similarity and/or sequence identity
with that of an original sequence or a portion of an original sequence
and may also have a modification of its structure as discussed herein.
The degree of similarity between two sequences is base upon the
percentage of identities (identical amino acids) and of conservative
substitution.

[0154] Similarity or identity may be compared, for example, over a region
of 2, 3, 4, 5, 10, 19, 20 amino acids or more (and any number
therebetween). Identity may include herein, amino acids which are
identical to the original peptide and which may occupy the same or
similar position when compared to the original polypeptide. An analog
which have, for example, 50% identity with an original polypeptide may
include for example, an analog comprising 50% of the amino acid sequence
of the original polypeptide and similarly with the other percentages. It
is to be understood herein that gaps may be found between the amino acids
of an analogs which are identical or similar to amino acids of the
original peptide. The gaps may include no amino acids, one or more amino
acids which are not identical or similar to the original peptide.
Biologically active analogs of the carriers (polypeptides) of the present
invention are encompassed herewith.

[0156] For example an analogue may comprise or have 50% identity with an
original amino acid sequence and a portion of the remaining amino acid
which occupies a similar position may be for example a non-conservative
or conservative amino acid substitution.

[0157] Therefore, analogues of the present invention comprises those which
may have at least 90% sequence similarity with an original sequence or a
portion of an original sequence. An "analogue" may have, for example t
least 35%, 50%, 60%, 70%, 80%, 90% or 95% (96%, 97%, 98%, 99% and 100%)
sequence similarity with an original sequence or a portion of an original
sequence. Also, an "analogue" may also have, for example, at least 35%,
50%, 60%, 70%, 80%, 90% or 95% (96%, 97%, 98%, 99% and 100%) sequence
similarity to an original sequence with a combination of one or more
modification in a backbone or side-chain of an amino acid, or an addition
of a group or another molecule, etc. Exemplary amino acids which are
intended to be similar (a conservative amino acid) to others are known in
the art and includes, for example, those listed in Table 1.

[0158] Analogues of the present invention also comprises those which may
have at least 35%, 50%, 60%, 70%, 80%, 90% or 95% (96%, 97%, 98%, 99% and
100%) sequence identity with an original sequence or a portion of an
original sequence. Also, an "analogue" may have, for example, 35%, 50%,
60%, 70%, 80%, 90% or 95% (sequence) identity to an original sequence
(i.e., an analogue that is at least 35%, 50%, 60%, 70%, 80%, 90% or 95%
identical to an original peptide) with a combination of one or more
modification in a backbone or side-chain of an amino acid, or an addition
of a group or another molecule, etc.

[0159] A "fragment" is to be understood herein as a polypeptide
originating from a portion of an original or parent sequence or from an
analogue of said parent sequence. Fragments encompass polypeptides having
truncations of one or more amino acids, wherein the truncation may
originate from the amino terminus (N-terminus), carboxy terminus
(C-terminus), or from the interior of the protein. A fragment may
comprise the same sequence as the corresponding portion of the original
sequence. Biologically active fragments of the carrier (polypeptide)
described herein are encompassed by the present invention.

[0160] Thus, biologically active polypeptides in the form of the original
polypeptides, fragments (modified or not), analogues (modified or not),
derivatives (modified or not), homologues, (modified or not) of the
carrier described herein are encompassed by the present invention.

[0161] Therefore, any polypeptide having a modification compared to an
original polypeptide which does not destroy significantly a desired
biological activity is encompassed herein. It is well known in the art,
that a number of modifications may be made to the polypeptides of the
present invention without deleteriously affecting their biological
activity. These modifications may, on the other hand, keep or increase
the biological activity of the original polypeptide or may optimize one
or more of the particularity (e.g. stability, bioavailability, etc.) of
the polypeptides of the present invention which, in some instance might
be needed or desirable. Polypeptides of the present invention comprises
for example, those containing amino acid sequences modified either by
natural processes, such as posttranslational processing, or by chemical
modification techniques which are known in the art. Modifications may
occur anywhere in a polypeptide including the polypeptide backbone, the
amino acid side-chains and the amino- or carboxy-terminus. It will be
appreciated that the same type of modification may be present in the same
or varying degrees at several sites in a given polypeptide. Also, a given
polypeptide may contain many types of modifications. Polypeptides may be
branched as a result of ubiquitination, and they may be cyclic, with or
without branching. Cyclic, branched and branched cyclic polypeptides may
result from posttranslational natural processes or may be made by
synthetic methods. Modifications comprise for example, without
limitation, pegylation, acetylation, acylation, addition of
acetomidomethyl (Acm) group, ADP-ribosylation, alkylation, amidation,
biotinylation, carbamoylation, carboxyethylation, esterification,
covalent attachment to fiavin, covalent attachment to a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative, covalent
attachment of drug, covalent attachment of a marker (e.g., fluorescent,
radioactive, etc.), covalent attachment of a lipid or lipid derivative,
covalent attachment of phosphatidylinositol, cross-linking, cyclization,
disulfide bond formation, demethylation, formation of covalent
cross-links, formation of cystine, formation of pyroglutamate,
formylation, gamma-carboxylation, glycosylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristoylation, oxidation,
proteolytic processing, phosphorylation, prenylation, racemization,
selenoylation, sulfation, transfer-RNA mediated addition of amino acids
to proteins such as arginylation and ubiquitination, etc. It is to be
understood herein that more than one modification to the polypeptides
described herein are encompassed by the present invention to the extent
that the biological activity is similar to the original (parent)
polypeptide.

[0162] As discussed above, polypeptide modification may comprise, for
example, amino acid insertion (i.e., addition), deletion and substitution
(i.e., replacement), either conservative or non-conservative (e.g.,
D-amino acids, desamino acids) in the polypeptide sequence where such
changes do not substantially alter the overall biological activity of the
polypeptide.

[0163] Example of substitutions may be those, which are conservative
(i.e., wherein a residue is replaced by another of the same general type
or group) or when wanted, non-conservative (i.e., wherein a residue is
replaced by an amino acid of another type). In addition, a non-naturally
occurring amino acid may substitute for a naturally occurring amino acid
(i.e., non-naturally occurring conservative amino acid substitution or a
non-naturally occurring non-conservative amino acid substitution).

[0164] As is understood, naturally occurring amino acids may be
sub-classified as acidic, basic, neutral and polar, or neutral and
non-polar. Furthermore, three of the encoded amino acids are aromatic. It
may be of use that encoded polypeptides differing from the determined
polypeptide of the present invention contain substituted codons for amino
acids, which are from the same type or group as that of the amino acid be
replaced. Thus, in some cases, the basic amino acids Lys, Arg and His may
be interchangeable; the acidic amino acids Asp and Glu may be
interchangeable; the neutral polar amino acids Ser, Thr, Cys, Gln, and
Asn may be interchangeable; the non-polar aliphatic amino acids Gly, Ala,
Val, Ile, and Leu are interchangeable but because of size Gly and Ala are
more closely related and Val, Ile and Leu are more closely related to
each other, and the aromatic amino acids Phe, Trp and Tyr may be
interchangeable.

[0165] It should be further noted that if the polypeptides are made
synthetically, substitutions by amino acids, which are not naturally
encoded by DNA (non-naturally occurring or unnatural amino acid) may also
be made.

[0166] A non-naturally occurring amino acid is to be understood herein as
an amino acid which is not naturally produced or found in a mammal. A
non-naturally occurring amino acid comprises a D-amino acid, an amino
acid having an acetylaminomethyl group attached to a sulfur atom of a
cysteine, a pegylated amino acid, etc. The inclusion of a non-naturally
occurring amino acid in a defined polypeptide sequence will therefore
generate a derivative of the original polypeptide. Non-naturally
occurring amino acids (residues) include also the omega amino acids of
the formula NH2(CH2)nCOOH wherein n is 2-6, neutral
nonpolar amino acids, such as sarcosine, t-butyl alanine, t-butyl
glycine, N-methyl isoleucine, norleucine, etc. Phenylglycine may
substitute for Trp, Tyr or Phe; citrulline and methionine sulfoxide are
neutral nonpolar, cysteic acid is acidic, and ornithine is basic. Proline
may be substituted with hydroxyproline and retain the conformation
conferring properties.

[0167] It is known in the art that analogues may be generated by
substitutional mutagenesis and retain the biological activity of the
polypeptides of the present invention. These analogues have at least one
amino acid residue in the protein molecule removed and a different
residue inserted in its place. Examples of substitutions identified as
"conservative substitutions" are shown in Table 1. If such substitutions
result in a change not desired, then other type of substitutions,
denominated "exemplary substitutions" in Table 1, or as further described
herein in reference to amino acid classes, are introduced and the
products screened.

[0168] In some cases it may be of interest to modify the biological
activity of a polypeptide by amino acid substitution, insertion, or
deletion. For example, modification of a polypeptide may result in an
increase in the polypeptide's biological activity, may modulate its
toxicity, may result in changes in bioavailability or in stability, or
may modulate its immunological activity or immunological identity.
Substantial modifications in function or immunological identity are
accomplished by selecting substitutions that differ significantly in
their effect on maintaining (a) the structure of the polypeptide backbone
in the area of the substitution, for example, as a sheet or helical
conformation. (b) the charge or hydrophobicity of the molecule at the
target site, or (c) the bulk of the side chain. Naturally occurring
residues are divided into groups based on common side chain properties:
[0169] (1) hydrophobic: norleucine, methionine (Met), Alanine (Ala),
Valine (Val), Leucine (Leu), Isoleucine (Ile), Histidine (His),
Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe), [0170] (2) neutral
hydrophilic: Cysteine (Cys), Serine (Ser), Threonine (Thr) [0171] (3)
acidic/negatively charged: Aspartic acid (Asp), Glutamic acid (Glu)
[0172] (4) basic: Asparagine (Asn), Glutamine (Gln), Histidine (His),
Lysine (Lys), Arginine (Arg) [0173] (5) residues that influence chain
orientation: Glycine (Gly), Proline (Pro); [0174] (6) aromatic:
Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe), Histidine (His),
[0175] (7) polar: Ser, Thr, Asn, Gln [0176] (8) basic positively charged:
Arg, Lys, His, and; [0177] (9) charged: Asp, Glu, Arg, Lys, His

[0178] Non-conservative substitutions will entail exchanging a member of
one of these classes for another. A conservative substitution will entail
exchanging a member of one of these groups for another member of these
groups. Alternatively other conservative amino acid substitutions are
listed in Table 1.

[0179] A biologically active analog may be, for example, an analogue
having at least one (i.e., non-conservative or conservative) amino acid
substitution in the original sequence. A biologically active analog may
also be for example, an analog having an insertion of one or more amino
acids.

[0180] Other exemplary analogs includes for example: [0181] A SEQ ID
NO.1 analog which may have the formula I: X1-SEQ ID NO.: 1-X2[0182] An Angiopep-1 analog which may have the formula II:
X1-Angiopep-1-X2 and [0183] An Angiopep-2 analog may have the
formula III: X1-Angiopep-2-X2

[0184] X1 and X2 may independently be an amino acid sequence of
from between 0 to about 100 (e.g., from between 0 to about 30 to 50)
amino acids. X1 and X2 may be derived from consecutive amino
acids of aprotinin or aprotinin analogs (homologous amino acid sequence)
or may be any other amino acid sequence (heterologous amino acid
sequence). A compound of either formula I, II or III may also comprises
an amino acid substitution, deletion or insertion within the amino acid
sequence of Angiopep-1, Angiopep-2 or SEQ ID NO.1. The analog however
would preferably be biologically active as determined by one of the
assays described herein or by any similar or equivalent assays.

[0185] A biologically active polypeptide (e.g., carrier) may be identified
by using one of the assays or methods described herein. For example a
candidate carrier may be produced by conventional peptide synthesis,
conjugated with Taxol as described herein and tested in an in vivo model
as described herein. A biologically active carrier may be identified, for
example, based on its efficacy to increase survival of an animal which
has been injected with tumor cells and treated with the conjugate
compared to a control which has not been treated with a conjugate. Also a
biologically active carrier may be identified based on its location in
the parenchyma in an in situ cerebral perfusion assay.

[0186] It is to be understood herein, that if a "range" or "group of
substances" is mentioned with respect to a particular characteristic
(e.g., temperature, concentration, time and the like) of the present
invention, the present invention relates to and explicitly incorporates
herein each and every specific member and combination of sub-ranges or
sub-groups therein whatsoever. Thus, any specified range or group is to
be understood as a shorthand way of referring to each and every member of
a range or group individually as well as each and every possible
sub-ranges or sub-groups encompassed therein; and similarly with respect
to any sub-ranges or sub-groups therein. Thus, for example, [0187] with
respect to a length of from 10 to 18 amino acid I, is to be understood as
specifically incorporating herein each and every individual length, e.g.,
a length of 18, 17, 15, 10, and any number therebetween etc.; Therefore,
unless specifically mentioned, every range mentioned herein is to be
understood as being inclusive. For example, in the expression from 5 to
10 amino acids long is to be as including 5 and 10; [0188] and similarly
with respect to other parameters such as sequences, length,
concentrations, elements, etc. . . .

[0189] It is in particular to be understood herein that the sequences,
regions, portions defined herein each include each and every individual
sequences, regions, portions described thereby as well as each and every
possible sub-sequences, sub-regions, sub-portions whether such
sub-sequences, sub-regions, sub-portions is defined as positively
including particular possibilities, as excluding particular possibilities
or a combination thereof; for example an exclusionary definition for a
region may read as follows: "provided that said polypeptide is no shorter
than 4, 5, 6, 7, 8 or 9 amino acids. Yet a further example of a negative
limitation is the following; a sequence comprising SEQ ID NO.: X with the
exclusion of a polypeptide of SEQ ID NO. Y; etc. An additional example of
a negative limitation is the following; provided that said polypeptide is
not (does not comprise or consist of) SEQ ID NO.:Z.

BRIEF DESCRIPTION OF THE DRAWINGS

[0190] In drawings which illustrates exemplary embodiments of the
invention,

[0198]FIG. 9 illustrates the effect of treatment of Taxol-Angiopep-2
conjugate on the survival of glioblastoma-implanted mice (athymic, nude
mice) and;

[0199]FIG. 9 illustrates the structure of exemplary polypeptides of the
present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0200] The present invention relates to new molecules that can act as
vectors or carriers for transporting an agent, medicine or other molecule
to the brain and/or central nervous system (CNS). Agents, medicines or
other molecules which are unable or ineffective at crossing the
blood-brain barrier by themselves, will be transported across the
blood-brain barrier when attached or coupled (conjugated) to the vector
or carrier. Alternatively, an agent that is able to cross the blood-brain
barrier by itself may also see its transport increase when conjugate to
the carrier of the present invention. Such conjugates can be in the form
of a composition, such as a pharmaceutical composition, for treatment of
a condition or disease.

[0201] Design of Candidate Molecules as Carrier Vectors

[0202] In international publication no. WO2004/060403, the inventors have
disclosed that AngioPep-1 (SEQ ID NO.:67) and aprotinin (SEQ ID NO.:98)
are effective vectors for transporting desirable molecules across the
blood brain barrier. The inventors herein demonstrate that other
molecules could also be used as carriers for transporting an agent across
the blood brain barrier. Accordingly, peptides having similar domains as
aprotinine and Angiopep-1 and a modified form of Angiopep-1 (amidated,
peptide no. 67) were therefore conceived as potential carrier vectors.
These derived peptides resemble aprotinine and Angiopep-1 but comprise
different amino acid insertions and bear different charges. Thus far, 96
peptides presented in Table 2 as well as additional peptides listed in
the sequence listing were tested for their potential as carrier.

[0203] It is to be understood herein that in the following experiments,
peptides have been selected based on their higher activity compared to
others. Those which have not been selected for further experimentations
are by no means being disclaimed and are not intended to be regarded as
non-functional. These peptides show substantial activity and have utility
has (biologically active) carriers and are also encompassed by the
present invention.

[0204] An in vitro model was used for screening assay and for mechanistic
studies of drug transport to the brain. This efficient in vitro model of
the blood-brain barrier was developed by the company CELLIAL®
Technologies. Yielding reproducible results, the in vitro model was used
for evaluating the capacity of different carriers to reach the brain. The
model consists of a co-culture of bovine brain capillary endothelial
cells and rat glial cells. It presents ultrastructural features
characteristic of brain endothelium including tight junctions, lack of
fenestration, lack of transendothelial channels, low permeability for
hydrophilic molecules and a high electrical resistance. Moreover, this
model has shown a good correlation coefficient between in vitro and in
vivo analysis of wide range of molecules tested. To date, all the data
obtained show that this BBB model closely mimics the in vivo situation by
reproducing some of the complexities of the cellular environment that
exist in vivo, while retaining the experimental advantages associated
with tissue culture. Many studies have validated this cell co-culture as
one of the most reproducible in vitro model of the BBB.

[0205] The in vitro model of BBB was established by using a co-culture of
BBCECs and astrocytes. Prior to cell culture, plate inserts (Millicell-PC
3.0 μM; 30-mm diameter) were coated on the upper side with rat tail
collagen. They were then set in six-well microplates containing the
astrocytes and BBCECs were plated on the upper side of the filters in 2
mL of co-culture medium. This BBCEC medium was changed three times a
week. Under these conditions, differentiated BBCECs formed a confluent
monolayer 7 days later. Experiments were performed between 5 and 7 days
after confluence was reached. The permeability coefficient for sucrose
was measured to verify the endothelial permeability.

[0208] Originally, at a first level of selection, 96 peptides as described
in Table 2 were tested as carrier with the in vitro model of the BBB.
Each peptide was added to the upper side of the inserts covered or
non-covered with endothelial cells for 90 minutes at 37° C. After
the incubation, the peptides in the lower side of the chambers were
resolved by electrophoresis. Electrophoresis gels were stained with
Coomassie blue to visualize the peptides as illustrated with some
peptides (without limitation) in FIG. 1. AngioPep-1 (either SEQ ID NO.:67
or peptide no. 67 (amidated form)) is often used herein as a reference or
for comparison purpose. In FIG. 1, each initial peptide applied to the
upper side of the filters was loaded on electrophoresis gel (ini) as
control. After 90 minutes of transcytosis, a volume of 50 μl from the
basolateral side of the filters covered with endothelial cells (+) or
non-covered (-) was also loaded on Tricine gels. To visualize the
peptides gels were stained with Coomassie blue.

[0209] Following the first level of screening, peptides detected in the
lower side of the chambers by Coomassie blue staining (5, 8, 45, 67, 70,
71, 72, 73, 74, 75, 76, 77, 78, 79, 81, 82, 90 and 91) were selected for
further study with the iodinated peptides. Briefly, the selected peptides
were iodinated with standard procedures using iodo-beads from Sigma. Two
iodo-beads were used for each protein. These beads were washed twice with
3 ml of phosphate buffer (PB) on a Whatman® filter and resuspended in
60 μl of PB. 125I (1 mCi) from Amersham-Pharmacia biotech was
added to the bead suspension for 5 min at room temperature. The
iodination for each peptide was initiated by adding 100 μg (80-100
μl) of the bead suspension. After an incubation of 10 min at room
temperature, the supernatants were applied on a desalting column
prepacked with 5 ml of cross-linked Dextran® from Pierce and
125I-proteins were eluted with 10 ml of PBS. Fractions of 0.5 ml
were collected and the radioactivity in 5 μl of each fraction was
measured. Fractions corresponding to 125I-proteins were pooled and
dialyzed against Ringer/Hepes buffer, pH 7.4. The efficiency of
radiolabeling was between 0.6-1.0×108 cpm/100 μg of
protein.

[0210] The iodinated peptides were also investigated with the in vitro
model of the BBB. Each peptide was added to upper side of the inserts
covered or non-covered with endothelial cells for 90 minutes at
37° C. After the incubation, peptides in the lower side of the
chambers were TCA precipitated. Results were expressed as cpm ratios. For
each [125I]-peptide the number of cpm in the bottom chamber was
divided by the total number of cpm added to filter covered with
endothelial cells (+cells/initial) or uncovered (-cells/initial). The
ratio between the number of [125I]-peptide found in the bottom
chamber of filters covered with or without endothelial cells was also
calculated (+cells/-cells). A very low -cells/initial ratio indicates
that filters may interfere with the peptides (peptides 5 and 8). A high
+cells/initial and +cells/-cells ratio indicate a better passage of the
peptides across the brain endothelial cells. The results for the
previously selected 18 peptides are shown in Table 3.

[0211] From these results, 12 peptides with +cells/-cells ratios generally
higher than 0.35 were selected namely; 5, 8, 67, 75, 76, 77, 78, 79, 81,
82, 90 and 91. Peptides #91 and #77 were also selected for further
investigation because of their +cells/-cells ratios (>0.2).

[0212] The 12 selected peptides were then investigated by assessing their
permeability coefficients using the in vitro BBB model. The effect of
each selected peptide at 250 nM on the BBB integrity was determined by
measuring [14C] sucrose permeability in the BBB model on BBCEC
monolayers grown on filters in the presence of astrocytes. To achieve
this test, brain endothelial cell monolayers grown on inserts were
transferred to 6-well plates containing 2 mL of Ringer-Hepes per well
(basolateral compartment) for two hours at 37° C. Ringer-Hepes
solution was composed of 150 mM NaCl, 5.2 mM KCl, 2.2 mM CaCl2, 0.2
mM MgCl2, 6 mM NaHCO3, 5 mM Hepes, 2.8 mM Hepes, pH 7.4. In
each apical chamber, the culture medium was replaced by 1 mL Ringer-Hepes
containing the labeled [14C]-sucrose. At different times, inserts
were placed into another well. [14C] sucrose passage was measured at
37° C., on filters without cells or with filters coated with BBCEC
cells. The peptides are added at the start of the experiment at time
zero. The results were plotted as the sucrose clearance (μl) as a
function of time (min).

[0216] The in situ cerebral perfusion (in mice) was used as the fourth
level of selection to select the best peptides. This procedure also
distinguishes between compounds remaining in the brain vascular
compartment from those having crossed the abluminal endothelial membrane
to enter the brain parenchyma. Indeed, the technique of post-perfusion
capillary depletion allows to measure whether the molecule really crosses
the endothelium to enter the brain parenchyma. Using this technique it is
demonstrated herein that specific peptides tend to accumulate in the
brain parenchyma fraction (see Table 4).

[0217] Four peptides, namely 5, 67, 76 and 91, showed the highest levels
of distribution in the parenchyma with a volume higher than 20 ml/100 g
and which represents at least 25% of the volume found for the total brain
(homogenate), thus showing the highest potential as carrier for use as
transport vectors. Peptide 79 was eliminated because of its lower volume
of distribution in the brain parenchyma (18 ml/100 g). Peptide 67
represents the amidated form of AngioPep-1 described in the previous
application that the inventors filed. Amidation of a peptide affect the
overall charge of the peptide. As is apparent in Tables 2 and 3, two
peptides having a different charge do not have necessary the same
activity.

[0218] The vector or carrier of the present invention may thus be used in
a method for transporting an agent across the blood-brain barrier
comprises administering to an individual an agent that comprises an
active ingredient or a pharmaceutical agent attached to a carrier, such
as aprotinin or a functional derivative thereof (i.e., an aprotinin
analog, an aprotinin fragment, an aprotin derivative, an analogue of an
aprotinin fragment).

[0219] The carrier and conjugate may be administered intra-arterially,
intra-nasally, intra-peritoneally, intravenously, intramuscularly,
sub-cutaneously, transdermally or per os to the patient. The agent may
be, for example, an anti-angiogenic compound. The agent may have a
maximum weight of 160,000 Daltons. As discussed herein, the agent may be
a marker or a drug such as a small molecule drug, a protein, a peptide or
an enzyme. The drug may be adapted to treat, for example, a neurological
disease or a central nervous system disorder of a patient. The drug may
be a cytotoxic drug and the marker may be a detectable label such as a
radioactive label, a green fluorescent protein, a histag protein or
β-galactosidase. The agent may be delivered, for example, into the
central nervous system of a patient.

[0220] According to another embodiment, the uses, methods, compounds,
agents, drugs or medicaments therein mentioned may not alter the
integrity of the blood-brain barrier of the patient.

[0221] According to a further embodiment of the present invention the
peptide may be selected from the group consisting of aprotinin, an
aprotinin fragment (SEQ ID NO.:1) and any one of the peptides defined in
SEQ ID NO.:1 to 97, 99, 100 or 101.

[0222] For example, peptides 5, 76, 91, 97 and 97 as well as peptide 67
may be used in the present invention by linking them to an agent or a
compound for transporting the agent or compound across the blood-brain
barrier of a patient. The agent or compound may be adapted to treat a
neurological disease or to treat a central nervous system disorder.

[0223] The carrier of the present invention, such as for example, peptides
5, 76, 91 and 97 as well as peptide 67 may be linked to or labelled with
a detectable label such as a radioimaging agent, such as those emitting
radiation, for detection of a disease or condition, for example by the
use of a radioimaging agent-antibody-carrier conjugate, wherein the
antibody binds to a disease or condition-specific antigen. Other binding
molecules besides antibodies and which are known and used in the art are
also contemplated by the present invention. Alternatively, the carrier or
functional derivative thereof of the present invention or mixtures
thereof may be linked to a therapeutic agent, to treat a disease or
condition, or may be linked to or labelled with mixtures thereof.
Treatment may be effected by administering a carrier-agent conjugate of
the present invention to an individual under conditions which allow
transport of the agent across the blood-brain barrier.

[0224] A therapeutic agent as used herein may be a drug, a medicine, an
agent emitting radiation, a cellular toxin (for example, a
chemotherapeutic agent) and/or biologically active fragment thereof,
and/or mixtures thereof to allow cell killing or it may be an agent to
treat, cure, alleviate, improve, diminish or inhibit a disease or
condition in an individual treated. A therapeutic agent may be a
synthetic product or a product of fungal, bacterial or other
microorganism, such as mycoplasma, viral etc., animal, such as reptile,
or plant origin. A therapeutic agent and/or biologically active fragment
thereof may be an enzymatically active agent and/or fragment thereof, or
may act by inhibiting or blocking an important and/or essential cellular
pathway or by competing with an important and/or essential naturally
occurring cellular component.

[0225] Examples of radioimaging agents emitting radiation (detectable
radio-labels) that may be suitable are exemplified by indium-111,
technitium-99, or low dose iodine-131.

[0226] Detectable labels, or markers, for use in the present invention may
be a radiolabel, a fluorescent label, a nuclear magnetic resonance active
label, a luminescent label, a chromophore label, a positron emitting
isotope for PET scanner, chemiluminescence label, or an enzymatic label.
Fluorescent labels include but are not limited to, green fluorescent
protein (GFP), fluorescein, and rhodamine. Chemiluminescence labels
include but are not limited to, luciferase and β-galactosidase.
Enzymatic labels include but are not limited to peroxidase and
phosphatase. A histag may also be a detectable label.

[0227] It is contemplated that an agent may be releasable from the carrier
after transport across the blood-brain barrier, for example by enzymatic
cleavage or breakage of a chemical bond between the carrier and the
agent. The release agent may then function in its intended capacity in
the absence of the carrier.

[0228] The present invention will be more readily understood by referring
to the following examples which are given to illustrate the invention
rather than to limit its scope. The following examples have been given
with aprotinin. However, it has been demonstrated herein the molecules of
the present invention share common properties with aprotinin with respect
to their potential as carrier for transporting an agent across the blood
brain barrier. These examples thus apply to the molecules of the present
invention.

Example I

Strategies for Drug Conjugation (Paclitaxel)

[0229] For conjugation, paclitaxel (TAXOL®) has 2 strategic positions
(position C2' and C7). FIG. 2 illustrates the method of attachment of the
vector or carrier of the present invention to paclitaxel. Briefly,
paclitaxel is reacted with anhydride succinic pyridine for 3 hours at
room temperature to attach a succinyl group in position 2'. Such
2'-succinyl paclitaxel has a cleavable ester bond in position 2' which
upon cleavage can simply release succinic acid. This cleavable ester bond
can be further used for various modifications with linkers, if desired.
The resulting 2'-O-succinyl-paclitaxel is then reacted with EDC/NHS in
DMSO for 9 hours at room temperature, followed by the addition of the
carrier or vector in Ringer/DMSO for an additional reaction time of 4
hours at room temperature. The reaction of conjugation depicted in FIG. 2
is monitored by HPLC. Each intermediate, such as paclitaxel,
2'-O-succinyl-paclitaxel and 2'-O--NHS-succinyl-paclitaxel, is purified
and validated using different approaches such as HPLC, thin liquid
chromatography, NMR (13C or 1H exchange), melting point, mass
spectrometry. The final conjugate is analyzed by mass spectrometry and
SDS-polyacrylamide gel electrophoresis. This allows determining the
number of paclitaxel molecules conjugated on each vector.

[0230] Transcytosis capacity of Aprotinin-Paclitaxel conjugate was
determined and is reported below in Table 5.

[0231] As seen in Table 5, conjugation of paclitaxel to aprotinin still
was able to cross the in vitro model of the blood brain barrier without
affecting the sucrose integrity, thus proving that the molecules (also
referred herein as vectors or carriers) of the present invention still
retain their activity when conjugated to a large chemical entity such as
paclitaxel.

[0232] Survival study in the rat brain tumor model was then conducted to
verify whether the paclitaxel that was conjugated is still active in
vivo. For the rat brain tumor model, rats received an intra-cerebral
implantation of 50 000 CNS-1 glioma cells. Three (3) days after, animals
received treatment with vehicle (aprotinin), Paclitaxel (5 mg/kg) or
Paclitaxel-Aprotinin (5 mg/kg) by intravenous injection. Treatment was
then administered every week until animal was sacrificed (see FIG. 3).
Rats were monitored every day for clinical symptoms and weight loss.
According to the protocol of good animal practice, animals were
sacrificed when a weight loss was observed for 3 consecutive days or
before if the weight loss was more than 20% of the animal initial weight.

[0234] Survival study was also conducted in mice implanted with a human
brain tumor xenograft. For the mice brain tumor model, mice received an
intra-cerebral implantation of 500 000 human U87 glioma cells. 3 days
after implantation animals received treatment with Paclitaxel-Angiopep1
(5 mg/kg) or vehicle by intravenous injection. Treatment was then
administered every week until animal was sacrificed. Mice were monitored
every day for clinical symptoms and weight loss. According to the
protocol of good animal practice, animals were sacrificed when a weight
loss was observed for 3 consecutive days or before if the weight loss was
more than 20% of the animal initial weight. It was now observed that the
medium survival for the control group was 19±2 days. For the
statistical analysis a 20% increase in survival was considered
significant. As can be seen in FIG. 4, the conjugate
Paclitaxel-AngioPep-1 retained its activity, having a statistically
significant effect. The survival time of the paclitaxel-angioPep1 treated
animals is significantly extended when compared to control group
(p<0.05, n=8).

[0235] Results obtained in the two survival studies indicate that the
conjugation of paclitaxel with the vector of the present invention
increases the animal survival.

Example II

Strategies for Antibodies Conjugation

[0236] Since proteins generally have several amino groups available for
conjugation, amine coupling using sulfa-NHS/EDC activation is be used to
cross-link therapeutic antibodies with the vectors (carriers) of the
present invention. This approach was chosen because it is a fast, simple
and reproducible coupling technique, because the resulting conjugate is
stable while still retaining the biological activity of the antibody and
it has a high conjugation capacity that can be reliably controlled and a
low non-specific interaction during the coupling procedures.

[0237] Antibodies or antibody fragments (Fab and Fab'2) have been
conjugated with the vector of the present invention to increase their
delivery to the brain. Various conjugation approaches have been used to
first conjugate IgGs with aprotinin, having proven that the carriers of
the present invention behave exactly as aprotinin.

[0238] Different cross-linkers, such as BS3
[Bis(sulfosuccinimidyl)suberate], NHS/EDC (N-hydroxysuccinimide and
N-ethyl-N'(dimethylaminopropyl)carbodimide or Sulfo-EMCS
([N-e-Maleimidocaproic acid]hydrazide) have been tested for the
conjugation of IgG. BS3 is a Homobifunctional N-hydroxysuccinimide
ester that targets accessible primary amines. NHS/EDC creates a
conjugation of primary amine groups with carboxyl groups. Sulfo-EMCS are
heterobifunctional reactive groups (maleimide and NHS-ester) that are
reactive toward: sulfhydryl and amino groups.

[0239] Conjugation of IgG with aprotinin using the cross-linker BS3
(FIG. 5) or sulfo-EMCS (FIG. 6) was first assessed.

[0240] Transport of IgG or IgG-conjugates across the BBB was then tested.
The uptake of [125I]-IgG to the luminal side of mouse brain
capillaries was measured using the in situ brain perfusion method adapted
in the inventor's laboratory for the study of drug uptake in the mouse
brain (Dagenais et al., 2000, J. Cereb. Blood Flow Metab. 20(2):381-386).
The BBB transport constants were determined as previously described by
Smith (1996, Pharm. Biotechnol. 8:285-307). IgG uptake was expressed as
the volume of distribution (Vd) from the following equation:

Vd=Q*br/C*pf

where Q*br is the calculated quantity of [125I]-IgG or
[125I]-IgG-aprotinin conjugate per gram of right brain hemisphere
and C*pf is the labelled tracer concentration measured in the perfusate.

[0241] The results of this experiment indicate that there is higher brain
uptake for [125I]-IgG-aprotinin conjugate than that of unconjugated
[125I]-IgG (see FIG. 7).

[0242] The conjugation of IgGs with aprotinin increases their accumulation
in the brain parenchyma in vivo.

Example III

Effect of Taxol-Angiopep-2 Conjugate on Mice Survival

[0243] This study with Taxol-Angiopep-2 (herein referred to peptide no. 97
(angiopep2 is not amidated) was conducted to determine whether
conjugation of Taxol to Angiopep-2 could increase mice survival. The
structure of Angiopep-2 is illustrated in SEQ ID NO.:97. For this
experiment, mice received an intra-cerebral implantation of 500 000 human
U87 glioma cells. After 3 days following implantation, animals were
treated with the vehicle (DMSO/Ringer-Hepes 80:20 v/v (i.e., control)) or
Taxol-Angiopep-2 conjugate (3:1, i.e., ratio of 3 Taxol molecules for
each peptide; TxlAn2 (5 mg/kg)) by tail vein injections (FIG. 8). Mice
were monitored every day for clinical symptoms and weight loss.
Treatments were administered until animals were sacrificed. As shown in
Table 6, we observed that the median survival was 18 days for the control
group whereas the median survival for mice receiving the Taxol-Angiopep-2
conjugate was 21 days (FIG. 8). Survival curve obtained for mice treated
with Taxol-Angiopep-2 conjugate (in red) indicates that the median
survival was significantly increased by 17% (FIG. 8). The statistical
analysis presented also in Table 6 indicates that administration of
Taxol-Angiopep-2 conjugate significantly increased survival by 17% (p
values=0.048).

[0244] The content of each publication, patent and patent application
mentioned in the present application is incorporated herein by reference.

[0245] Although the present invention has been described in details herein
and illustrated in the accompanying drawings, it is to be understood that
the invention is not limited to the embodiments described herein and that
various changes and modifications may be effected without departing from
the scope or spirit of the present invention.

[0246] While the invention has been described in connection with specific
embodiments thereof, it will be understood that it is capable of further
modifications and this application is intended to cover any variations,
uses, or adaptations of the invention following, in general, the
principles of the invention and including such departures from the
present disclosure as come within known or customary practice within the
art to which the invention pertains and as may be applied to the
essential features hereinbefore set forth, and as follows in the scope of
the appended claims.